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First published online 12 December 2007
doi: 10.1242/dev.015081

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An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors

¹ Department of Anatomy and Program in Developmental Biology, School of Medicine, University of California at San Francisco, San Francisco, CA 94143-2711, USA.
² Department of Pediatrics, School of Medicine, University of California at San Francisco, San Francisco, CA 94143-2711, USA.
³ Department of Orofacial Sciences, School of Dentistry, University of California at San Francisco, San Francisco, CA 94143-0758, USA.
⁴ Department of Preventive and Restorative Dental Sciences, School of Dentistry, University of California at San Francisco, San Francisco, CA 94143-0758, USA.
⁵ Department of Craniofacial Development, King's College London, London, SE1 9RT, UK.
⁶ Department of Teratology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

View larger version (91K): [in this window] [in a new window]   Fig. 1. Spry4-/-;Spry2+/- mice develop a `tusk'-like incisor due to the presence of enamel on the lingual surface. (A) Schematic diagram of an adult incisor. Enamel and dentin, the calcified tissues of the tooth, are produced by ameloblasts and odontoblasts, respectively. Note that in the normal incisor, ameloblasts and enamel are present only on the labial surface. (B,F) Side views of mandibles from wild-type and Spry4-/-;Spry2+/- adult mice with soft tissue removed. Note the abnormal length and thickness (red bar) of the mutant incisor, as well as the absence of a sharp tip (asterisk). (C-E) Coronal sections from an XTM analysis of wild-type, Spry4-/- (4-/-), and Spry4-/-;Spry2+/- (4-/-;2+/-) incisors. The colors in the bar on the left indicate mineral content. In the XTM sections, enamel is false-colored blue and purple, and dentin and bone are false-colored green and yellow. The white arrow points to ectopic enamel. (G,H) Sagittal sections of the mandibular incisor from postnatal day 14 wild-type and Spry4-/-;Spry2+/- animals. Here, and in all other panels in this and subsequent figures, anterior is to the left and posterior to the right. Yellow arrowheads point to a layer of enamel. Black arrows point to the CLs at the posterior end of the lingual and labial sides of the incisor. (I-L) Higher magnification views of regions boxed in G and H. Note that in the wild-type and mutant incisors, dentin (stained pink and/or blue) and odontoblasts are present on both lingual and labial sides, whereas enamel and ameloblasts are found only on the labial side in wild-type and on both labial and lingual sides in the Spry4-/-;Spry2+/- incisor. (M-P) High magnification views showing lingual and labial CL morphology in wild-type and Spry4-/-;Spry2+/- incisors. Note that the mutant lingual CL is more similar to the labial CL than to the wild-type lingual CL. Scale bars: 500 µm in H; 100 µm in L; 50 µm in P. A, anterior; Am, ameloblasts; CL, cervical loop; De, Dentin; En, enamel; Ep, epithelium; Mes, mesenchyme; M1 and M2, first and second molar; Od, odontoblasts; P, posterior; wt, wild type.

View larger version (85K): [in this window] [in a new window]   Fig. 2. Shh expression reveals the presence of ectopic pre-ameloblasts in Spry4-/-;Spry2+/-, in Spry4-/-, and in Fst-/- embryonic mouse incisors. (A-I) Gene expression analyzed by RNA in situ hybridization using the indicated probes on paraffin sections of wild-type and mutant embryonic incisors. The stage at which the embryos were collected is indicated. In this and subsequent figures, a dotted line outlines the incisor epithelium. In D, the Shh expression domain appears to extend slightly into the lingual epithelium, but as this was not reproducibly observed it is likely to be an artifact of the plane of section. The red arrowheads in E and H point to a domain in which Shh is ectopically expressed on the lingual side of Spry4-/-;Spry2+/- and Spry4-/- incisors. A similar domain is detected in Fst null incisors (panel I), together with an additional anterior domain of ectopic Shh expression (open arrowheads). Scale bars: 100 µm.

View larger version (133K): [in this window] [in a new window]   Fig. 3. FGF genes are upregulated or ectopically expressed on the lingual side of Spry4-/-;Spry2+/- and Spry4-/- embryonic incisors. (A-K) Gene expression was analyzed by RNA in situ hybridization using the probes indicated on paraffin sections of E16.5 incisors of the genotypes denoted. Yellow asterisks indicate gene expression domains in the mesenchyme, and arrows point to the anterior and posterior ends of gene expression domains in the epithelium. All samples are shown at the same magnification.

View larger version (129K): [in this window] [in a new window]   Fig. 4. Loss of Spry4 function alone is not sufficient to maintain continuous production of ameloblasts on the lingual side of the incisor. (A-C) Sagittal section of postnatal day (P) 5 Spry4-/- incisor. The areas boxed in A are shown at higher magnification in B and C. Note that unlike on the labial side, the lingual ameloblast layer does not extend to the posterior end of the incisor. (D-G) RNA in situ hybridization assays for Shh and Fgf3 expression in paraffin sections of P2 incisors of the genotypes indicated. Yellow asterisks indicate mesenchymal expression, and black arrows demarcate the extent of epithelial expression in these panels. Note that no ectopic expression of either gene is detected in the Spry4-/- incisors at this stage. (H) Frequency of the tusk phenotype (presence of lingual enamel) in Spry4-/-;Spry2+/- adult mice, and the effects of reducing the dosage of Fgf9 or Fgf10. The Spry4-/-;Spry2+/- animals evaluated were pooled from both crosses used to generate animals with reduced FGF gene dosage. P-values were calculated using Fisher's exact test. Am, ameloblasts; Od, odontoblasts.

View larger version (109K): [in this window] [in a new window]   Fig. 5. sprouty gene expression and tissue-specific inactivation in the developing mouse incisor. Gene expression was analyzed by RNA in situ hybridization using the probes indicated on paraffin sections of embryonic incisors of the genotypes denoted at E16.5 or 17.5. (A-F) A comparison of the expression domains of sprouty gene family members in the incisor at the stages indicated. Yellow asterisks indicate mesenchymal expression. (G-K) Tissue-specific inactivation of Spry4. The absence of Spry4 expression is indicated by red circles in the epithelium of an incisor carrying K14-cre, one Spry4fl and one Spry4- allele (G) and by red asterisks in the mesenchyme of an incisor carrying Wnt1-cre and two Spry4fl alleles (H). For each genotype shown in I-K, the diagram illustrates the tissue in which Cre-mediated recombination occurred (green fill), the sprouty alleles that were inactivated by Cre (white lettering), or that were inherited as nulls (black lettering). The photograph shows Shh expression, which marks cells that are differentiating along the ameloblast lineage. The red arrowheads point to the ectopic lingual Shh expression domain.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Models for the role of sprouty genes in controlling FGF signaling in the mouse incisor and for the generation of embryonic ameloblasts. (A) Functions of sprouty genes in inhibiting the establishment of a lingual FGF epithelial-mesenchymal signaling loop. Arrows indicate a stimulatory effect and the symbol indicates an inhibitory effect of one signaling molecule on the expression of another. In wild type, sprouty genes are expressed on the labial side, but they do not prevent (dashed symbol) reciprocal signaling between FGF9 in epithelium and FGF3/FGF10 in mesenchyme. On the lingual side, sprouty genes inhibit signaling to adjacent tissues by the low levels of FGF9 in the epithelium and of FGF10 in the mesenchyme, and consequently there is no upregulation of FGF gene expression in either tissue. However, in Spry4 null incisors a reciprocal signaling loop between epithelium and mesenchyme is established because, in the absence of SPRY4, these tissues are hypersensitive to the low level of FGF signaling. In turn, the increase in FGF signaling on the lingual side results in the generation of ameloblasts from self-renewing stem cells in the CL. (B) A proposal for how ameloblasts develop in the embryonic incisor. At E15.5, wild-type incisor epithelium contains embryonic ameloblast progenitor (EAP) cells capable of limited proliferation. On the labial side, their descendants (in the domain colored pink) differentiate `in situ' into enamel-producing cells. Similar cells are present on the lingual side (in the domain colored lighter pink), but their differentiation is inhibited by Follistatin. Between E15.5 and 16.5, an ameloblast stem cell (ASC) population is established in the labial CL. Unlike EAP cells, ASCs have the capacity to self-renew (circular arrow), as well as give rise to ameloblasts. ASC descendants that will develop into enamel-producing cells may first form transit-amplifying (T-A) cells in the anterior CL. After several divisions, their descendants move out of the CL, and begin differentiating. In the E16.5 incisor, these ASC-derived pre-ameloblasts are found in a domain in the labial epithelium (colored dark brown), between the EAP domain and the CL. The diagram illustrates the possibility that ASCs are normally also present on the lingual side. However, no pre-ameloblasts derived from these ASCs are present in the lingual epithelium anterior to the CL (in the domain colored light brown), because the generation of ameloblasts from lingual ASCs is blocked due to the inhibitory effects of sprouty as well as FST function on lingual FGF gene expression.

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